Polymerase chain-reaction-mediated cloning and expression of the coat protein gene of potato virus Y in Escherichia coli.
Complementary DNAs (cDNAs) to the RNA genome of a necrotic strain of potato virus Y in Japan (Hokkaido Univ. isolate of PVY-T:PVY-TH) were synthesized and cloned into a plasmid pBR322. About 4.3 kbp of the cDNA sequence containing the 3'-poly(A) tract of PVY-TH was inserted into a recombinant plasmid pBRYT88. The coat protein coding region (CP gene) in pBRYT88 was amplified using the polymerase chain reaction (PCR) and subcloned into a plasmid pUC119. The nucleotide sequence of the CP gene was determined from both the PCR-mediated clones and pBRYT88. The CP gene of PVY-TH consisted of 801 nucleotides, corresponding to 267 amino acids of Mr 29,811. The predicted amino acid sequence of the PVY-TH CP gene was different from that of PVYN (1) in only five amino acids and displayed 98.1% sequence homology. This result indicates that PVY-TH is closely related to PVYN (1). The cDNAs of the PVY-TH CP gene containing an additional initiation codon (ATG) at the 5' end and a stop codon at the 3' end were constructed by PCR amplification and subcloned into an E. coli expression vector, pKK223-3. Five transformed E. coli colonies expressing the PVY-TH CP were identified by immunoscreening using both polyclonal rabbit antiserum against PVY-TH and mouse monoclonal antibody (MoAb) specific to PVY-T. The CP of PVY-TH produced in the E. coli colonies had an electrophoretic mobility identical to that of native PVY-TH CP and reacted strongly to a specific MoAb to PVY-T, but did not react to a specific MoAb to an ordinary strain of PVY (PVY-O). The maximum expression of the CP in E. coli was approximately 7% of the total soluble proteins. The result indicates that the CP gene cloned by PCR was functional and the PCR procedure was useful for producing biologically active cDNA clones from a single, long positive-sense RNA genome encoding a single, large polyprotein precursor, such as potyviruses.
Hataya
T
T
Biotechnology Business Division, Nippon Steel Co., Ltd., Tokyo, Japan.
Sano
T
T
Ohshima
K
K
Shikata
E
E
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002213:Capsid / Q000235:genetics*; D003001:Cloning, Molecular; D015246:Deoxyribonuclease EcoRI / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005814:Genes, Viral; D008969:Molecular Sequence Data; D010942:Plant Viruses / Q000235:genetics*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D011198:Solanum tuberosum
D015246:Deoxyribonuclease EcoRI